www.nature.com/scientificreports OPEN Morphological and molecular analysis of cryptic native and invasive freshwater snails in Chile Received: 20 August 2018 Gonzalo A. Collado1,2, Marcela A. Vidal1,2, Karina P. Aguayo1, Marco A. Méndez 3, Accepted: 29 January 2019 Moisés A. Valladares 3, Francisco J. Cabrera 4, Luis Pastenes5, Diego E. Gutiérrez Gregoric6,7 & Published online: 24 May 2019 Nicolas Puillandre8 Species delimitation in minute freshwater snails is often difcult to perform using solely shell morphology. The problem intensifes when invasive species spread within the distribution range of morphologically similar native species. In Chile, the Truncatelloidean snails are represented by the native genera Heleobia and Potamolithus plus the invasive mudsnail Potamopyrgus antipodarum, which can easily be confused. Using an integrative approach, we performed molecular phylogenetic analysis and studied reproductive and morphological features to identify superfcially similar forms inhabiting the central area of the country. Truncatelloidean snails were identifed in 40 of 51 localities sampled, 10 containing Potamopyrgus antipodarum, 23 Heleobia and 7 Potamolithus. Based on these results and previously published data, the known distribution of the mudsnail in Chile encompasses 6 hydrological basins, including 18 freshwater ecosystems. The fnding of the mudsnails in several type localities of native species/subspecies of “Heleobia” that were not fnd in situ suggests species replacement or signifcant extinction of native fauna, a hypothesis supported by the restudy of type material that shows that endemic forms belong to the genus Potamolithus. This study shows the usefulness of integrative taxonomy not only resolving complex taxa with cryptic morphology but also measuring the extent of an ongoing invasion. Non-native species can generate a range of efects on ecosystems invaded. One of the most serious impacts on the environment is the threat to endemic biodiversity, which may be afected through population demise, rarefaction, species displacement and even extinction1–7. In some cases, biological invasions may be difcult to recognize due to the introduction of cryptic species8, a problem that intensifes when invasive species colonize ecosystems located within the known distribution range of morphologically similar native species, which may lead, in turn, to unfortunate misidentifcations9. The gastropods of the superfamily Truncatelloidea include minute freshwater snails that are difficult to identify due to small size, substantial intraspecifc variability and convergence of external shell features9–12. In Chile, these snails are represented by two native genera: Heleobia Stimpson, 1865 (Cochliopidae), with about 30 species, and Potamolithus Pilsbry, 1896 (Tateidae species), composed by Potamolithus australis Biese, 1944 and Potamolithus santiagensis (Biese, 1944)13–22. A third representative is the mudsnail Potamopyrgus antipodarum (Gray, 1843) (Tateidae), native to New Zealand23,24, a highly invasive species the morphology of which closely resembles snails of the two Chilean endemic genera25,26. Besides these taxa, the mudsnail can also be confused with other Tateidae as well as other species belonging to diferent gastropod families, including Cochliopidae, Amnicolidae, Bithyniidae, Hydrobiidae, and Lithoglyphidae27–32. 1Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad del Bío-Bío, Chillán, Chile. 2Grupo de Biodiversidad y Cambio Global, Universidad del Bío-Bío, Chillán, Chile. 3Laboratorio de Genética y Evolución, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. 4Universidad Tecnológica de Chile INACAP, Chillán, Chile. 5Departamento de Biología y Química, Facultad de Ciencias Básicas, Universidad Católica del Maule, Talca, Chile. 6División Zoología Invertebrados, Museo de La Plata, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina. 7Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET), CCT La Plata, Buenos Aires, Argentina. 8Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d’Histoire naturelle, CNRS, Sorbonne Université, EPHE, 57 rue Cuvier, CP 26, 75005, Paris, France. Correspondence and requests for materials should be addressed to G.A.C. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:7846 | https://doi.org/10.1038/s41598-019-41279-x 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Te morphological similarity of the mudsnail with native snails has led to misidentifcations in invaded habi- tats. For instance, in Garden Lakes, USA, the native Tryonia porrecta (Mighels, 1845) was incorrectly identifed as the mudsnail31 whereas in Nordic areas from Europe, some species of Bithynia Leach, 1818 are also superfcially similar in morphology to this species29. Te mudsnail also resembles the snail Pyrgophorus platyrachis Tompson, 1968, native to Florida and subsequently introduced in Singapore and the Middle East9,11,12. Identifcation errors can even lead to the description of a cryptic invasive species as a new species, making the correct identifcation of the invasive species, and consequently, the invasion, overlooked25,33,34. Perhaps one of the more striking exam- ples of this occurred at the end of the 19th century when the mudsnail was described from the Tames estu- ary (UK) as Potamopyrgus jenkinsi (Smith, 1889), an overlooked biological invasion that was fnally confrmed almost a century later23,33,35. Similarly, in Australia and Tasmania the mudsnail was also erroneously recognized as Potamopyrgus niger (Quoy and Gaimard, 1835) for a long time33. In Chile, the presence of the mudsnail was frst reported in 2014 and later in 2016 but the species was introduced earlier since it was confused with native species of the genus Heleobia in 201118,25,26. Accurate identifcation of freshwater snails in the Southern Cone of South America becomes even more difcult considering that native species of Potamolithus have been confused with Heleobia18,22,36–38, for example “Heleobia” hatcheri (Pilsbry, 1911) and “Heleobia” santiagensis. The invasive populations of the mudsnail can reach very high densities in the invaded ecosystems39–43. Investigations of the efect of this species on these environments have shown both positive and negative rela- tionships with the invertebrate taxa44. For example, some studies have demonstrated the absence of signifcant changes in local communities with respect to biomass and biodiversity, as well as decrease in population abun- dances and local extinctions of some species22,41,45–49. However, other studies have found an increase in densities of macroinvertebrates, as well as an increase in family richness and diversity44,50,51. Molecular analyses based on DNA sequences are a powerful tool for accurate identifcation of invasive spe- cies52,53, including freshwater snails25,31,34,54–56. Tese studies can be coupled to traditional morphological inves- tigations in an integrative way. Te objective of this study was to identify cryptic native and invasive snails of the superfamily Truncatelloidea distributed in central Chile, including samples collected from six type localities of native species, using molecular and morphological analysis. We also reexamined the type specimens of the taxa originally described under the genus Littoridina Souleyet, 185210,13–18 in the study area to investigate if this repre- sents original misidentifcations or possible extinctions of native fauna. Results Taxa identifcation. Of 51 localities sampled, the preliminary morphological examination identifed trun- catelloidean snails in 40 of them (Table S1). Te shell, superfcially similar among populations (Fig. 1a,d,g), precluded an objective and prompt identifcation of taxa in the feld. A close examination of the protoconch (Fig. 1b,e,h) and operculum (Fig. 1c,f,i) allowed us to diferentiate the mudsnail from Heleobia and Potamolithus snails and also to distinguish between these two endemic genera, but with greater difculty. Te protoconch showed diferences in length among genera, being longer in the mudsnail. Te operculum, corneous, paucispiral and with eccentric nucleus in all populations studied, showed diferences among genera regarding the color and the presence/absence of a white smear (Fig. 2a). Reproductive features showed some particular diferences in second- ary characters, sexuality and reproductive strategies that also allowed diferentiating among genera (Fig. 2b–h). In all populations examined the radula is taenioglossan (Fig. 3a–f), with seven teeth on each row, including two marginal teeth (external and internal) plus a lateral tooth placed on each side of the central (rachidian) tooth (Fig. 3a,c,e); the general formula is 3–1–3. Tis structure allowed identifying snails to the family level according to diferences provided by the central and external marginal teeth. A summary with the main morphological diferences of the samples studied are shown in Table 1. Specimens of Potamopyrgus antipodarum were found in 10 sites, Heleobia spp. in 23 and Potamolithus spp. in 7. In no locality were all three genera found co-distributed. In all the localities where Heleobia occurs, no other truncatelloidean snail was found. Potamopyrgus antipodarum and Potamolithus live in syntopy in two localities. Heleobia populations were not identifed at the species level due to the variability of morphological characters, the fnding of putative new species and the fact that only mudsnails were